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Yokohama-shi, Japan

Matsuda A.,University of Tokyo | Matsuda A.,Japan Science and Technology Agency | Ogawa M.,Osaka University | Yanai H.,University of Tokyo | And 10 more authors.
Biochemical and Biophysical Research Communications | Year: 2011

The activation of innate immune responses is critical to host defense against microbial infections, wherein nucleic acid-sensing pattern recognition receptors recognize DNA or RNA from viruses or bacteria and activate downstream signaling pathways. In a search for new DNA-sensing molecules that regulate innate immune responses, we identified RNA-binding motif protein 3 (RBM3), whose role has been implicated in the regulation of cell growth. In this study, we generated Rbm3-deficient (Rbm3-/-) mice to study the role of RBM3 in immune responses and cell growth. Despite evidence for its interaction with immunogenic DNA in a cell, no overt phenotypic abnormalities were found in cells from Rbm3-/- mice for the DNA-mediated induction of cytokine genes. Interestingly, however, Rbm3-/- mouse embryonic fibroblasts (MEFs) showed poorer proliferation rates as compared to control MEFs. Further cell cycle analysis revealed that Rbm3-/- MEFs have markedly increased number of G2-phase cells, suggesting a hitherto unknown role of RBM3 in the G2-phase control. Thus, these mutant mice and cells may provide new tools with which to study the mechanisms underlying the regulation of cell cycle and oncogenesis. © 2011.

Okada O.,ZoeGene Corporation | Odai K.,Shohoku College | Sugimoto T.,Kanto Gakuin University | Ito E.,Tokushima Bunri University
Biophysical Chemistry | Year: 2012

The gating of ion channel of ionotropic glutamate receptors is controlled by the structural change of the ligand-binding domain of GluR2. We examined the roles of residues in the glutamate-binding and cleft-closing mechanisms by molecular dynamics (MD) simulations. A glutamate entered the cleft deeply within the order of nanoseconds and the cleft locked the glutamate completely at 15 ns in an MD run. TYR450 seemed to regulate the orientation of the glutamate upon binding by cation-π interaction. A semi-open state was identified in the free energy profile evaluated with the structures on the spontaneously glutamate-bound and cleft-closed pathway by the unbiased MD simulations for the first time to our knowledge. In the semi-open state, the two sub-domains were bridged by two hydrogen bonds of GLU705 in the sub-domain 2 with TYR732 in the sub-domain 1 and with the glutamate bound to the sub-domain 1 until the transition to the closed state. © 2011 Elsevier B.V. All rights reserved.

Ogata K.,ZoeGene Corporation | Ogata K.,RIKEN | Shen J.-W.,Mitsubishi Group | Shen J.-W.,Chiyoda Corporation | And 5 more authors.
Bulletin of the Chemical Society of Japan | Year: 2012

Molecular dynamics (MD) simulations were used to investigate the dissociation of the Ras/Raf complex. Three models of Ras/Raf complexes were used in this study: two active GTP-bound forms with either GTP or GDP and one inactive GDP-bound form. The dissociation process in these short time scale (10 ns) MD simulations was accelerated by adding random velocities to phosphate groups in the GTP or GDP molecule. Only the complex with the inactive GDPbound form heated with the random velocities showed dissociation. The Raf in it was unable to recover its initial position during the MD simulations. In simulations with and without added random velocities the Raf in the other complexes moved back to its initial position. The Raf in complexes with the active GTP-bound form recovered its initial position even when the ligands in the complexes were heated. The details of these mechanisms are discussed here. © 2012 The Chemical Society of Japan.

Kobayashi T.,ZoeGene Corporation | Kobayashi T.,Molecuence Corporation | Kakui M.,ZoeGene Corporation | Shibui T.,ZoeGene Corporation | And 2 more authors.
Molecular Biotechnology | Year: 2011

Interleukin-6 (IL-6) plays a crucial role in malignant diseases, such as rheumatoid arthritis, Castleman disease, and multiple myeloma, and as such, is an attractive therapeutic target. Here, the authors isolated a novel IL-6 inhibitor peptide by in vitro selection using mRNA display. The authors first used a random-primed human cDNA library to isolate IL-6-binding peptides. After four rounds of selection, a 19-amino acid peptide named CA11 was selected and confirmed to specifically interact with IL-6. The authors then performed an alanine scan analysis of CA11 and determined the amino acid residues necessary to interact with IL-6. Next, the authors constructed a CA11-based partially randomized library and after ten more rounds of selection, isolated several groups of peptides. The most frequently occurring sequence, RA07, bound to IL-6 with 3 to 4-fold higher affinity than CA11. Furthermore, RA07 inhibited IL-6-dependent KT-3 cell proliferation in a dose-dependent manner. ELISAs revealed that RA07 could not inhibit IL-6 from binding to the IL-6 receptor (IL-6R), but could inhibit the IL-6/IL-6 complex binding to gp130. © 2010 Springer Science+Business Media, LLC.

Kaji T.,ZoeGene Corporation | Yoshida S.,ZoeGene Corporation | Kawai K.,ZoeGene Corporation | Fuchigami Y.,ZoeGene Corporation | And 3 more authors.
Biochemical and Biophysical Research Communications | Year: 2010

Apoptosis signal-regulating kinase 1 (ASK1) and ASK2 are both members of mitogen-activated protein kinase kinase kinase (MAP3K) family that are implicated in apoptotic cell death, stress responses, and various diseases. We have determined that NT2RI3007443, TESTI4031745, SGK341, and human MAP3K15 are all transcribed from the same genomic locus, which we designate "ASK3 gene" based on sequence homology to ASK1 and ASK2. NT2RI3007443, TESTI4031745, and SGK341 displayed distinct expression profiles among human tissues. TESTI4031745 was expressed in relatively high levels. The expression of TESTI4031745 was increased in rectum tumor and Alzheimer's disease hippocampus and decreased in kidney tumor and Alzheimer's disease frontal lobe. NT2RI3007443 showed moderate levels of ubiquitous expression in normal adult tissues. They did not drastically change in diseases except for increase in cirrhosis liver. Expression of SGK341 was restricted. It was highly expressed in fetal brain, and moderately expressed in normal hippocampus, pancreas, spleen, lung, and kidney. Further, its expression was dramatically increased in hepatic cirrhosis and decreased in lung tumor. Target proteins encoded by NT2RI3007443 and TESTI4031745 were translated in cell-free protein synthesis system. They exhibited protein kinase activity indicated by ATP consumption and phosphorylation of Syntide 2 as a substrate. We demonstrated that knockdown of ASK3 protected HeLa cells against cytotoxicity induced by anti-Fas monoclonal antibody, TNF-alpha, or oxidative stress. These findings suggest that "ASK3 gene" is a novel member of apoptosis signal-regulating kinases and that it plays a pivotal role in the signal transduction pathway implicated in apoptotic cell death triggered by cellular stresses. It can be a putative therapeutic drug target for multiple human diseases. © 2010 Elsevier Inc. All rights reserved.

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